HIV-2 peptides

ABSTRACT

A substantially pure polypeptide having at least one antigenic determinant that is substantially identical to an antigenic determinant of a protein from a cell line infected with simian T-lymphotrophic virus-III or human T-lymphotrophic virus-IV (HTLV-IV), also known as HIV-2, the protein being selected from: a) a glycoprotein having a molecular weight (m.w.) of about, 160,000 daltons; a glycoprotein having a m.w. of about 120,000 daltons; a gag protein having a m.w. of about 55,000 daltons; a gag protein having a m.w. of about 24,000 daltons; and a glycoprotein having a m.w. of about 32,000 daltons. Also disclosed are various methods of immunoassay using that peptide or antibodies raised to it. Finally, immunoassays for simian specimens are disclosed using peptides that are immunologically cross-reactive with the above-described peptide, or antibodies thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of, and is a continuation of,application Ser. No. 08/378,872, filed Jan. 24, 1995 (pending), which isa continuation of application Ser. No. 07/984,231, filed Dec. 1, 1992(abandoned), which is a continuation of application Ser. No. 07/658,460,filed Feb. 22, 1991 (abandoned), which is a continuation of applicationSer. No. 07/538,680, filed Jun. 15, 1990 (abandoned), which is acontinuation of application Ser. No. 06/844,072, filed Mar. 26, 1986(abandoned), which is a continuation-in-part of application Ser. No.06/798,126, filed Nov. 14, 1985 (abandoned).

This invention was made with Government support, including NIH researchgrants CA37466 and CA18216, and National Research Service Award ST32CA9382. The Government has certain rights in the invention. Thisapplication is a continuation-in-part of our commonly owned pendingapplication Ser. No. 798,126, filed Nov. 14, 1985, which is herebyincorporation by reference.

BACKGROUND OF THE INVENTION

This invention relates to primate T-lymphotrophic viruses, as well asassays for such viruses and substances used in those assays.

A group of closely related human retroviruses that preferentially infecthelper T-lymphocytes have been designated human T-lymphotrophic viruses(HTLV). One type of HTLV, designated HTLV-I, has been linked with thedevelopment of adult T-cell leukemia/lymphoma (Poiesz et al. (1980)Proc. Nat'l. Acad. Sci. USA 77:7415). A virus related to HTLV-I has beenreported in non-human primates, specifically Asian and African Old Worldprimate species, but not New World primates and prosimians. The primateviruses from baboons, African green monkeys, and Macaca species arerelated to, yet distinct from, HTLV-I. Guo et al. (1984) Science223:1195; Tsujimoto et al. (1985) Virology 144:59.

Another type of HTLV, designated variously as HTLV-III, orLymphadenopathy Associated Virus (“LAV” or “ARV”) is the prototype virusfrom patients with acquired immune deficiency syndrome (AIDS) (Popovicet al. (1984) Science 224:497; Salahuddin et al. (1984) Science 224:500;Schupbach et al. (1984) Science 224:503; Sarngadharn et al. (1984)Science 224:506). Various antigenic proteins from HTLV-III infectedcells have been repported, including:

1) a 55 kd gag polyprotein (p55) which yields a 24 kd protein (p24) asthe major virus core protein, and a 17 kd phosphoprotein (pp17)(Schupbach et al. (1984) Science 224:503-505); and

2) an envelope glycoprotein (gp160) which gives rise to a 120 kdglycoprotein (gp120) at its amino terminus (Essex and Lee, U.S. Ser. No.670,361, filed Nov. 9, 1984, and a continuation-in-part thereof filedNov. 7, 1985, both of which are hereby incorporated by reference).

SUMMARY OF THE INVENTION

We have discovered an exogenous type C retrovirus that infects simianspecies and is closely related to HTLV-III. Specifically, cells infectedwoth simian T-lymphotrophic virus-III (STLV-III) produce proteins thatare generally immunologically cross-reactive with the respective majorproteins produced by HTLV-III infected cells. STLV-III infects Africangreen monkeys (AGM), and Macaca species, and may infect other primatespecies. As used in this application, the term African green monkeysinclude all animals classified as members of genus Cercopithecus andparticularly the species C. aethiops. Growth characteristics, T-4tropism, and ultrastructural morphology of STLV-III are similar to thatof HTLV-III. The STLV-III that infects Macaca species (STLV-III_(MAC))induces biological effects similar to those that HTLV-III induces inhumans, including immunodeficiency or immunosuppressive disease. TheSTLV-III that infects African green monkeys (STLV-III_(AGM)) does notappear to produce disease. The term STLV-III is used in this applicationto include STLV-III_(AGM) and any all forms, subtypes and variations ofthose and other HTLV-III-like retroviruses that infect simians.

We have also discovered a human virus, HTLV-IV, that is virtuallyindistinguishable from STLV-III_(AGM) by immunological techniques, and,like STLV_(AGM), does not appear to cause AIDS or ARC-like symptoms ininfected humans. The term HTLV-IV is used to designate viruses thatimmunologically are more closely related to STLV-III than to HTLV-III asindicated by the strength and breadth (number of determinantsrecognized) of immunological cross reactivity. The term HTLV-IV is usedfor convenience to refer to human viruses, without necessarily implyingany distinction between STLV-III and HTLV-IV.

The discovery and characterization of STLV-III and HTLV-IV is importantin several respects. First, STLV-III- and HTLV-IV-infected cells providea source of antigenic determinants that are generally useful in assaysof simian or human specimens, as described below. Second, the animalspecies particularly at risk for STLV-III infection, the African greenmonkeys, is used for research and development of a variety of biologicalreagents; for example African green monkey tissue is used in theproduction of oral polio vaccine. It is desirable to reduce the chance(however unlikely) that an AIDS-like disease could be transmittedinadvertantly in polio vaccine or other products produced fromSTLV-III-infected animal tissue. Third, since STLV-III_(AGM) and HTLV-IVdo not appear to cause disease in infected monkeys or humans, yet areimmunologically cross-reactive with disease-causing HTLV-III, a vaccinebased on STLV-III or HTLV-IV could protect against AIDS.

Peptides having STLV-III or HTLV-IV antigenic determinants and assaysusing them

Accordingly, a first aspect of the invention generally features asubstantially pure polypeptide having at least one antigenic determinantthat is substantially identical to an antigenic determinant of a proteinfrom a cell line infected with STLV-III or HTLV-IV, the protein beingselected from: a) a glycoprotein having a molecular weight (m.w.) ofabout 160,000 daltons; a glycoprotein having a m.w. of about 120,000daltons; a gag protein having a m.w. of about 55,000 daltons; a gagprotein having a m.w. of about 24,000 daltons; and a glycoprotein havinga m.w. of about 32,000. By “a polypeptide having an antigenicdeterminant that is substantially identical to a protein antigenicdeterminant” is meant a polypeptide comprising an antigenic determinantwhich: a) in common with the protein antigenic determinant, will reactwith a given antibody; and b) is derived either by i) isolating thenaturally produced protein or a fragment of it; or ii) synthesizing(e.g. by expression of DNA such as by the general method of Chang et al.(1985) Nature 315:151, or chemical synthesis) an amino acid sequenceidentical to the protein antigenic determinant. As demonstrated below,the STLV-III and HTLV-IV cell proteins are immunologicallycross-reactive with HTLV-III cell proteins, but the reaction of anSTLV-III or HTLV-IV protein with a given antibody may vary in comparisonto the reaction of the corresponding HTLV-III protein with the sameantibody. Therefore, while the STLV-III and HTLV-IV antigenicdeterminants may be substantially identical for purposes of thisapplication, neither STLV-III nor HTLV-IV determinants are substantiallyidentical to HTLV-III determinants.

Preferably, the polypeptide antigenic determinant is substantiallyidentical to an antigenic determinant of a protein expressed in a cellline infected with STLV-III_(AGM) or HTLV-IV. Also preferably, thepolypeptide is one of the proteins listed above, or a fragment thereof;most preferably, the polypeptide is a gp32 or a gp160 or gp120glycoprotein in the glycosylated or unglycosylated form. Alsopreferably, the polypeptide is not substantially cross-reactive with theHTLV-III/LAV glycoprotein p41; and the polypeptide antigenic determinantis more strongly reactive with a determinant of an STLV-III or HTLV-IVglycoprotein than with an HTLV-III glyprotein determinant. Other usefulpolypeptides which have the necessary immunogenic determinants includesynthetic polypeptides.

The above described polypeptides of the first aspect of the inventionare useful, among other things, for assaying for the pressure ofantibodies to T-lymphotrophic viral antigens, by incubating a specimenwith the polypeptide and determining whether or not an immunocomplex isformed. Also, the above-described polypeptides can be used to raise anantibody that is useful for assaying a biological specimen (e.g., humanor simian) for the presence of an antigenic determinant that isimmunologically cross-reactive with a determinant of one of the fourproteins listed above. The assay is performed by incubating the specimenwith the antibody thus raised and determining whether an immunocomplexis formed. The determinants to be assayed may occur on the statedproteins themselves or on other polypeptides. They may be in freecirculation in the body fluids or in lymphocytes. The assay can becarried out by known immunoassay methods, using antibodies, monoclonalor polyvalent, having immune reactivity with the antigenic determinantsfound on the stated proteins. For example, competitive immunoassays orimmunometric (sandwich) assays can be used. The assays of the firstaspect are preferably performed on simian specimens, but they can alsobe performed on human specimens.

Assays of simian specimens

While the first aspect of the invention features assays that may beconducted on human as well as simian specimens, there is a second aspectof the invention specifically featuring assays on simian specimens. Inthis aspect, the assay for antibodies to viral antigens can be performedas described above, but a broader class of polypeptides can be used.Thus the second aspect is not limited to assays for antibodies usingpolypeptides that have an antigenic determinant that is “substantiallyidentical” to the proteins as defined above; the assays use anypolypeptide with an antigenic determinant that is immunologicallycross-reactive with a determinant of one of the five listed STLV-III orHTLV-IV proteins, regardless of whether the polypeptide determinant is“substantially identical” to the protein determinant.The polypeptidesused in the assays of the second aspect include those obtained from celllines infected with HTLV-III, HTLV-IV, or STLV-III (most preferablySTLV-III_(AGM) or HTLV-IV). The polypeptides used in the assays of thesecond aspect include the glycoproteins (in their glycosylated orunglycosylated form) described in Essex and Lee, U.S. Ser. No. 607,361,filed Nov. 9, 1984 which is hereby incorporated by reference. It hasbeen shown that anti-idiotypic reagents are useful for the detection ofantigens carrying sites which are immunologically cross-reactive withthose on the antibodies (Potocnjak et al. Science (1982) 215:1637-1639).Thus, such anti-idiotypic antibodies (or immunologically activefragments of them) which can be used as an assay for the presence ofantibodies to STLV-III viral antigens; specifically it inlcludesantibodies or fragments thereof which are anti-idiotypic towards theactive determinants of STLV-III or HTLV-IV infected cell proteins. Suchan anti-idiotypic antibody can be raised against antibodies to theproteins. Preferably, monoclonal antibodies are used.

Also, the second aspect of the invention features assaying simianspecimens for an antigenic determinant of a protein of STLV-III asgenerally described above by incubating the specimen with an antibody(preferably a monoclonal antibody) raised to a polypeptide that isimmunologically cross-reactive with one of the five proteins of STLV-IIIor HTLV-IV infected cells. Preferably, the antibody is raised to proteinof a cell infected with STLV-III or HTLV-IV, specifically to gp32,gp120, or gp160 of such a cell.

Vaccines

Finally, in a third aspect, the invention features a vaccine comprisingSTLV-III_(AGM), HTLV-IV, or a subunit protein or polypeptide therof,such as the gp120, or gp160 or a peptide fraction of those moleculesthat reacts with HTLV-III, This could be presented in a pharmaceuticallyacceptable carrier. Vaccine could also comprise proteins from cellsinfected with STLV-III_(AGM) or HTLV-IV, or an altered form thereof.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiment and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENT

We first briefly describe the drawings.

I. Drawings

FIGS. 1-8 are photographs demonstrating immunoprecipitation of variousserum samples with preparations of radiolabeled STLV-III or HTLV-IVinfected cell lysates with SDS/PAGE.

FIG. 9 is a photograph demonstrating Western blot analysis of STLV-IIIand HTLV-IV.

II. Obtaining the proteins

The proteins are isolated from cell lines infected with STLV-III orHTLV-IV. The reference STLV-III and HTLV-IV cell lines were derived fromco-cultivation of infected lymphocytes and HUT-78 cells. The HUT 78 cellline is a well-characterized mature human T-cell line reported in Gazdaret al. Blood 55:409 (1980); Poiesz et al. Proc. Nat'l. Acad. Sci. USA77:6815 (1980). Two such HUT-78/STLV-III_(AGM) cell lines have beendeposited with the American Type Culture Collection under ATCC Nos. CRL8942, 8943. An HTLV-IV/HUT-78 cell line is deposited with the ATCC asVR2129.

The preferred proteins are the gp160/120 glycoproteins, which have amolecular weight of approximately 120,000 daltons and 160,000 daltons asdetermined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS/PAGE), gel electrophoresis, and the proteins are soluble in SDSbuffer consisting of 0.15 M sodium chloride, 0.05 M Tris hydrochloridepH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodiumdodecylsulfate, and 1 mM phenylmethylsulfonyl fluoride. Triton X-100 isa nonionic detergent (octylphenoxy polyehtoxy (9-10) ethanol). Theunglycosylated moiety of the 160,000 dalton glycoprotein has a molecularweight of approximately 90,000 daltons and is immunogenic, sharing someantigenic determinant or determinants with the glycoproteins themselves.

A variety of other cell lines can be infected with STLV-III or HTLV-IV;among them can be mentioned H9 cells, NC37 cells, Molt 3 cells, Molt 4cells, and CEN cells. It may be that the exact sizes of the novelglycoproteins are slightly different in different lines; however, thecommon immunologically cross-reactive portion of the glycoproteins isthe same regardless of cell line, since it is a protein induced bySTLV-III or HTLV-IV. Thus, any cell which harbors the virus may be anappropriate source for the novel glycoproteins.

In order to obtain the protein from any infected cells carrying thevirus, the cells are metabolically labelled (e.g. with ³⁵S-cysteine) andimmunoprecipitated with antisera obtained from STLV-III infected animalsor HTLV-IV infected humans. The glycoproteins can be prepared withlentil-lectin affinity chromatography from infected cell lysate andsubjected to SDS/PAGE. For example, the glycoproteins are present incell HUT 78/STLV-III- or HTLV-IV-infected cell lines. HUT 78 cells canbe infected with fresh virus isolates from infected animals or humans.The glycoproteins can readily be separated from the cells of these celllines by lysis thereof and SDS gel electrophoresis.

III. Assays

Purified and isolated glyproteins or any antigen immunologicallycross-reactive therewith can be employed as a standard antigen in anyconventional assay procedure for detection of antibodies specificthereto in biological specimens, and hence detection of the presence inthe specimen of cells infected with STLV-III or HTLV-IV.

The glycoproteins or polypeptides immunologically cross-reactivetherewith can be labelled by conventional procedures with ¹²⁵I or ³⁵S or³H for use in radioimmunoassay, with fluorescein for fluorescentimmunoassay, with enzyme for enzyme immunoassay, or with biotin forbiotin-avidin inked assays. It can be employed labelled or unlabelled asdesired, in competitive immunoassays, as well as in double antibodyassays using two antibodies, either of the idiotype:antiidiotype varietyor more particularly of the second antibody type using an anti-Fcantibody, or other assays.

Alternatively, the glycoproteins or polypeptides immunologicallycross-reaction therewith could be immobilized on an insoluble phase,such as an insoluble resin, and detection of the anti-glycoproteinantibodies is carried out by measuring their binding to the insolublephase. Insoluble phases also include latex particles, which, when coatedwith the novel glyprotein or its immunologically cross-reactionpolypeptides and subjected to anti-glycoprotein antibody, willagglutinate. Yet other insoluble phases include test tubes, vials,titration wells, and the like, to which the novel glycoprotein or itsimmunologically cross-reactive polypeptide can be bound, and antibodythereto detected by double antibody techniques or Protein-A dependenttechniques.

The assay for antibodies which recognizes STLV-III- or HTLV-IV-inducedcell surface antigens may utilize the glycoprotein or glycoproteins orthe unglycosylated moiety of MW 120,000 daltons, 160,000 daltons and90,000 daltons respectively in crude form, and is not limited to usingthese proteins in substantially pure form. For example, theglycoprotein(s) may be first substantially purified and then mixedtogether. Alternatively cruder mixtures can also be used.

The elements necessary for carrying out the diagnostic methodologydescribed hereinbefore may be present in a kit. Such kit comprises acarrier being compartmentalized to receive therein one or morecontainers, which of said containers comprising one or more elementsnecessary to carry out the tests.

For example, the first container may contain one or both of the purifiedglycoproteins or its immunologically cross-reactive polypeptides indetectably labelled or in insolubilized form.

A second container may comprise anti IgG antibody, polyclonal ormonoclonal, useful in double antibody binding assay, or elements neededfor detection of the label on the glycoprotein or its immunogicallycross-reactive polypeptides (e.g. chromogenic substrates).

Additional containers may comprise varying amounts of one of theglycoproteins or its immunologically cross-reactive polypeptides whichcan be used to prepare a standard curve into which experimental resultscan be interpolated. The materials may be present in the kit bythemselves, in solution, freeze-dried, or in admixture with other insertmaterials, such as inert proteins, and the like.

The biological specimens tested may include blood, serum, lymphocytes,urine, tissues, saliva, feces, and the like. Of particular interest isthe screening of blood or other tissue of African green monkeys to beused in vaccine production.

The following specific examples are intended to illustrate more fullythe nature of the invention withou acting as a limitation upon itsscope.

EXAMPLES 1 AND 2: ASSAY OF STLV-INFECTED MACAQUE SERUM WITH STLV-IIIANTIGENS

FIG. 1 shows the reactivity of various simian serum samples with theglycoprotein preparation of HUT-78/STLV-III_(MAC) (lane a) anduninfected control HUT-78 (lane b). The glycoproteins are prepared fromsoluble cell lysates of the cells made with RIPA buffer as describedbelow for FIG. 2, lacking sodium deoxycholate. The lysates are passedthrough a lentil—lectin—Sepharose Cl-4B (Pharmacia, Sweden) column at aratio of 20×10⁶ cells to 1 to 2 ml of undiluted lentil—lectin—SepharoseCl-4B. The glycoproteins are eluted from the column with a buffer [0.15M NaCl, 0.05 M tris-HCl, pH 7.2, 1% Triton X-100, and 0.2methyl-α-D-mannoside]. The eluted bound fraction is reacted with 10 μltest sera that have been reacted to protein A beads (for example used inthe general technique of Essex et al. [1983] Science 220:859). The seraused are: Lanes 1 and 2-human reference HTLV-III, positive serum; Lane3--human reference HTLV-III, negative serum; Lanes 4 and 5--STLV-IIIpositive macaque serum; Lane 6--STLV-III negative macaque serum. Theimmunoprecipitates are eluted from protein A beads boiling at 100° C.for 2 min. [sample buffer: 0.1 Cleland's reagent; 2% SDS; 0.08 Mtris-HCl; pH 6.8; 10 percent glycerol; 0.2% bromophenol blue.] Samplesare analyzed by SDS/PAGE in a 10.0 percent acrylamide resolving gel witha 3.5 percent stacking gel according to the discontinuous buffer systemof Laemnili (1970) Nature (London) 227:680.

In FIG. 2, HUT-78/STLV-III_(MAC) (lanes a) and HUT-78 (lanes b) areharvested at their peak of log-phase growth and are exposed to [³⁵S]cysteine [150 μCi/ml; specific activity, 1000 to 1050 Ci/mmole; NewEngland Nuclear] for 8-10 hours. A soluble cell lysate is prepared bydisruption of cells with RIPA buffer (0.15 M NaCl, 0.5 tris-HCl, pH 7.2,1% sodium deoxycholate, 0.1% SDS). The lysate is then centrifuged for 1h. at 100,000 g. The lysate are reacted with 10 μl of the following testsera: lane 1--monoclonal anti-p24 (HTLV-III); lanes 2 and 3--humanreference HTLV-III, positive serum; lane 4--human reference HTLV-III,negative serum; lanes 5 and 6--macaque reference STLV-III positiveserum; lane 7--macaque reference STLV-III negative serum.

In both FIGS. 1 and 2, the STLV-III-infected macaque serum recognizesthe gp120/gp160 protein of STLV-III_(MAC)-infected cells.

Example 3: Macaca STLV-III as determined by HTLV-III infected cells

As shown in FIG. 3, the procedures described in example 2 were performedwith HTLV-III-infected H9 cells using at lanes 1-5 the sera describedfor FIG. 2 at lanes 1-5 and using a representative macaque serumnegative for STLV-III at lane 6.

Examples 4-6: STLV-III detected in Simians by STLV-III cell proteins

In FIG. 4, the above-described procedures were performed using HUT78/STLV-III(a) and HUT 78(b) on the following serum samples: lane 1)monoclonal anti-p24 (HTLV-III); lane 2) human reference serum positiveto HTLV-III; lane 3) human reference serum negative to HTLV-III; lane 4)macaque serum positive for STLV-III; lanes 5) and 6) AGM sera, positivefor STLV-III; lane 7) AGM negative for STLV-III; lane 8) baboon serumnegative for STLV-III; lane 9) chimpanzee serum negative for STLV-III.The 160, 120, 55, and 24 kd viral antigens of STLV-III areimmunoprecipitated by positive macaque sera and by HTLV-III positivesera.

FIG. 5 demonstrates protein expression in seven different African greenmonkeys that are infected with STLV-III. Cell cultures from thosemonkeys were derived by cocultivation with HUT 78 cells. The cellcultures were metabolically labeled with ³⁵S cysteine and RIP-SDS/PAGEof whole cell lysates was performed with: a) an STLV-III antibodypositive AGM serum sample; b) an STLV-III antibody negative AGM serumsample; and c) and HTLV-III antibody positive reference serum samplefrom a human with AIDS-related complex. Lanes designated C represent HUT78 uninfected cells that were similarly labeled.

Specifically, cells from the above-described cultures were exposed to[³⁵S] cysteine [−150 Ci/ml; specific activity 1000-1050 Ci/mmole; NewEngland Nuclear (NEN)] for four to six hours. A soluble cell lysate wasprepared by disrupting cells with RIPA buffer (0.15 M NaCl, 0.05 MTris-HCl, pH7.2, 1% sodium deoxycholate, and 0.1% SDS), and clearing bycentrifugation for one hour at 100,000 g. Each group of cell lysateswere reacted with 10 μl of the test sera bound to Protein A-sepharoseCL-4B (Proteins A-beads, Sigma). The immunoprecipitates were eluted in asample buffer containing 0.1 M Cleland's reagent, 2% SDS, 0.08 MTris-HCl, pH 6.8, 10% glycerol, and 0.2% bromophenol blue by boiling at100° C. for two minutes. Samples were analyzed in a 10.0% acrylamideresolving gel with 3.5% stacking gel according to the discontinuousbuffer system of Laemmli (1970) Nature 227:680

As shown in FIG. 5, lysates from all seven cultures (lanes 1-7) showedbands of about 160 kd, 120 kd, 55 kd, and 24 kd, when reacted with serumfrom a reference antibody positive African green monkey. The same bandswere not seen with lysate from uninfected HUT 78 cells (lane C). Theproteins were also not detected when lysates from the seven infectedcell cultures were reacted with a representative serum from an Africangreen monkey that lacked antibodies to STLV-III. Serum from a human ARCpatient recognized the 55 kd and 24 kd proteins and had faint reactivityto proteins of about 120 kd and 160 kd in lysates prepared from isolates1-7 but the same bands were lacking uninfected HUT 78 cells.

FIG. 6 shows immunoprecipitation by serum samples from the same sevendifferent African green monkeys from which virus was successfullyisolated were reacted with cell lysates prepared from an STLV-III_(AGM)cell line (isolate 1) and uninfected HUT 78 cells (lanes 1-7).STLV-III_(AGM) (isolate 1) infected (lanes a) and HUT 78 uninfected(lanes b) cell lysates were prepared as described above. Lanes H⁺contain serum samples from HTLV-III antibody positive human AIDS or ARCpatients; lane H⁻ contains a health control antibody negative humanserum; lanes 1-7 are serum samples from African green monkeys from whichvirus isolates 1-7 were obtained; lanes 8-12 are serum samples fromrepresentative African green monkeys that were positive for antibodiesto STLV-III_(MAC), and lane 13 is a representative African green monkeyserum that was negative for antibodies to STLV-III_(MAC).

As shown in FIG. 6, sera from virus positive monkeys specificallyprecipitated the gp160/120, whereas only three of these serum samplesadditionally showed reactivity to the p55 and p24. Analysis of otherAfrican green monkeys sera has demonstrated a similar phenomenon where11 of 42 (26%) STLV-III_(AGM) positive serum samples showed reactivityto th e p55 and p24 in addition to reactivity to the gp160/120. Thus,the high molecular weight glycoproteins of this virus apparently are themost immunogenic species in infected monkeys. STLV-III_(AGM) positivesera from African green monkeys were also analyzed for antibodies toHTLV/III/LAV proteins by RIP/SDS-PAGE where approximately 50% of thesesera also showed reactivity to HTLV-III proteins. Other diagnosticassays to detect this cross-reactivity reaction, in which the same serumsamples were analyzed by ELISA to HTLV-III (Electronucleonics).STLV-III_(AGM) virus and antibody positive monkeys demonstratedantibodies to HTLV-III proteins by RIP SDS/PADE in 5 of 8 serum sampleswhereas ELISA detected 2 of these 8 samples as HTLV-III positive. Thus,it appears that the presence of antibodies to STLV-III specific proteinswas the most closely associated with virus isolation. RIP and SDS/PAGEanalysis appear to be more sensitive for the detection of thecross-reacting antibodies to the related virus in infected monkeys whencompared to the HTLV-III kit ELISA.

Example 7: Reactivity of HTLV-IV with HTLV-III and STLV-III

Serum samples from healthy individuals (prostitutes or surgery patients)were obtained in Dakar, Senegal, and screened by a commericallyavailable HTLV-III/LAV ELISA kit assay. ELISA-positive samples were thenanalyzed by RIP/SDS/PAGE as generally described above, to eliminatefalse positives.

All positive samples demonstrated strong reactivity to all of theSTLV-III viral antigens, including p24, p55, and gp120/gp160. Only 27%of the samples demonstrated reactivity to all HTLV-III/LAV antigens;some of the samples failed to demonstrate any detectable antibodies toany major HTLV-III/LAV antigens, while others possessed antibodies onlyto p24 and p55 gag-related antigens. In all cases the reactivity toHTLV-III gp120 and gp160 was weak or non-existent compared to reactivityto STLV-III gp120/160. And there was a lack of detectable antibodies togp41 when analyzed by Western blot analysis by the general technique ofBarin et al. Lancet, ii, pp. 1387 et seq. (1985).

Representative serum samples from these West African individuals areshown in FIG. 7, with control serum samples from STLV-IIIantibody-positive African green monkeys. Specifically, serum samplesfrom people residing in West Africa were analyzed by RIP-SDS/PAGE usingthe whole cell lysates as follows. Cells from HTLV-III (BH10 virus)infected Molt-3 cells, uninfected Molt-3 cells, Hut-78 infectedSTLV-III_(AGM), and uninfected Hut-78 cells were harvested at their peakof log phase of growth and were exposed to (³⁵S) cysteine (−150Ci/ml;specific activity 1000-1050 Ci/mmol; New England Nuclear (NEW) for 4-6hours. A soluble cell lysate was prepared by disrupting cells with RIPAbuffer (0.15 M NaCl, 0.05 M Tris-HCl, pH 7.2, 1% sodium deoxycholate,and 0.1% SDS), and clearing by centrifugation for 1 hour at 100,000 g.Each group of cell lysates were reacted with 10 μl of the following testsera bound to Protein A-Sepharose Cl-4B (Protein A-beads, Sigma); (Lanes1-2) STLV-III_(AGM) antibody positive African green monkeys; (lane 3)healthy West African control with antibodies to STLV-III_(AGM); (lanes4-8) sera from West African prostitutes with reactivity toSTLV-III_(AGM); (lane 9) STLV-III_(AGM) and HTLV-III seronegativeprostitute from West Africa; and (lane 10) healthy West African controlseronegative for STLV-III_(AGM) and HTLV-III.

Immunoprecipitates were eluted in a sample buffer containing 0.1 MCleland's reagent, 2% SDS, 0.08 M Tris-HCl, pH 6.8, 10% glycerol, and0.2% bromophenol blue by boiling at 100′ for 2 mintues. Samples wereanalyzed in a 10.0% acrylamide resolving gel with 3.5% stacking gelaccording to the discontinuous buffer system of Laemmli referencedabove.

Example 8: Isolation of HTLV-IV

To isolate HTLV-IV, peripheral blood lymphocytes were obtained fromeight STLV-III_(AGM) antibody positive people and were cocultivated withHut-78 cells. The procedures for virus isolation was generally the sameas that described above for STLV-III_(AGM) or in Kanki et al. (1985)Science 230:951. Remarkably, the in vitro cytolysis of target cells wasnot observed. Therefore, Hut-78 cells were only added once to eachculture at day 5. At 21 to 28 days in culture, cellular atypia andmultinucleated giant cells were evident. Begining at 14 days afterinitiation, all cell cultures were monitored for viral proteinexpression by membrane immunofluoroescence (MIF) and RIP-SDS/PAGE aspreviously described, using a battery of reference sera with knownantibody reactivity to STLV-III_(AGM), HTLV-III/LAV, and HTLV-I viralproteins. After 28-35 days in culture, viral proteins related toSTLV-III were detected in 3 cultures by both MIF and RIP-SDS/PAGE.

Cell-free supernatants from the 3 cultures were monitored for Mg+dependent reverse transcriptase as previously described by Popovic etal. (1984) Science 224:497 and Salahudin et al. (1984) Science 224:500.3,500-93,000 counts per minute (cpm) over background (76-860 cpm) wasobserved in the 3 cell cultures expressing STLV-III viral proteins andnot in cultures that were negative for viral proteins.

Cell cultures expressing antigens cross reactive with STLV-III wereexamined by electron microscopy. Particles characteristic of aretrovirus were observed budding from infected cell membranes.Extracellular virions demonstrated an electron-dense cylindrical core,similar to that described for both simian and human T-lymphotrophicviruses. The ultrastructural morphology of retroviral particles seen inall three cultures derived from STLV-III antibody positive people ofWest Africa were similar to the retroviral particles of a referenceSTLV-III_(AGM) cell line derived from an African green monkey. It isnotable that the spike proteins of STLV-III type virions from bothmonkey and human origin were more prominent than those usually obserbedwith HTLV-III/LAV virions. The level of virus production from the WestAfrican STLV-III related virus culture was similar to that observed withHTLV-III/LAV infected H9 cells, based on both visual examination andreverse transcriptase activity.

Example 9: Identification of Antigens of HTLV-IV

As shown in FIG. 8, whole cell lysates from three HTLV-IV cell cultures(lanes 1-3), STLV-III_(AGM) reference infected Hut-78 (S) and uninfectedHut-78 cells (C) were prepared as described above and analyzed byRIP-SDS/PAGE as follows. Each group of cell lysates were reacted withthe following test sera: Negative serum sample from West Africa, virusand antibody negative for STLV-III and HTLV-III, STLV-III antibodypositive serum from individual 1, HTLV-III antibody positive serum fromindividual 2, STLV-III antibody positive serum from individual 3,reference STLV-III antibody positive serum from an African green monkey,and reference HTLV-III antibody positive serum from an AIDS patient.

Serum from a negative control individual that lacked antibodies to bothSTLV-III and HTLV-III/LAV failed to recognize any specific proteins inany of the five lysates. Conversely, lysates from STLV-III-positivecultures 1, 2, and 3 demonstrated bands of about 160, 129, 55 and 24 kdwhen reacted with their own sera or serum from a reference STLV-IIIantibody positive African green monkey (lanes 1, 2, and 3). These bandswere indistinguishable from proteins with similar electrophoreticmobility precipitated from reference STLV-III_(AGM) whole cel lysate(lanes S). These bands were not detectable when the same sera reactedwith uninfected Hut-78 cell lysates (lane C) or similarly prepared wholecell lysates from cultures derived from antibody negative people fromWest Africa. Serum from a reference U.S. AIDS patient recognized the 55and 24 kd proteins of STLV-III_(AGM) and reacted similarly with the sameproteins in cultures 1-3; only faint reactivity to the high molecularweight proteins, gp120/160, was observed.

As shown in FIG. 9, the viral antigens of STLV-III_(AGM) recognized bythe Western blot procedure include the p24, p15, p53, p64, gp120 andgp32. p24 and p15 are gag-related and analogous to similar viralproteins of HTLV-III/LAV. A 120 kd protein has been demonstrated withsome STLV-III and HTLV-IV antibody positive serum samples; this proteinis though to be analogous to the HTLV-III/LAV gp120 which is lessfrequently detected by Western blot procedures but readily detected byHTLV-III/LAV antibody positive samples by RIP-SDS/PAGE. The smearingband at 32 kd using the Western blot technique of Barin et al. Lancetii, p. 1387 (1987) correlates with a similar appearing 32 kdglycoprotein observed with lentil-lectin preparations and RIP-SDS/PAGE.The p32 protein may represent the transmembrane glycoprotein ofSTLV-III_(AGM) and HTLV-IV, by analogy with the gp41 of HTLV-III/LAV.The p64 and p53 of STLV-III_(AGM) and HTLV-IV are analogous to two polgene products of HTLV-III/LAV, p53 and p64.

Western blots can be performed as demonstrated by the following example.Cell-free virus from culture 1-3 as well as STLV-III_(AGM) virus from areference cell line was collected from supernatant fluid and subjectedto Western blotting by the general technique of Barin et al. Strips wereincubated with the same serum samples described in connection with FIG.8. STLV-III_(AGM) antibody positive reference serum from an Africangreen monkey showed reactivity with gp32, p24, p53, and p64 ofSTLV-III_(AGM) as well as similar bands in the three virus preparationfrom prostitutes from West Africa (FIG. 9). Similar proteins wererecognized using serum samples from individuals that had yieldedSTLV-III related viruses (HTLV-IV) with some variability in recognitionto the gp120. Control antibody negative serum failed to detect thesebands in any of the four virus preparations.

As shown in FIG. 9, Western blot analysis was performed on viruspreparations of the 3 HTLV-IV cell cultures (lanes 1-3), and anSTLV-III_(AGM) reference infected cell line (S). Each group of stripswere reacted with: Negative serum sample from West Africa, virus andantibody negative for STLV-III and HTLV-III, STLV-III antibody positiveserum from individual 1, STLV-III antibody positive serum fromindividual 2, STLV-III antibody positive serum from individual 3, andreference STLV-III antibody positive serum from an African green monkey.

IV. Vaccine

Since STLV-III_(AGM) and HTLV-IV are immunologically cross-reactive withHTLV-III, and yet present data indicates reduced (or no) pathenogenicityin their respective hosts. STLV-III_(AGM), HTLV-IV or a portion orderivative thereof could be used as a vaccine to protect againstHTLV-III.

Specifically, using tryptic peptide analysis of the STLV-III_(AGM) orHTLV-IV peptide antigens, it is possible to determine the antigenicdeterminants which cross react with HTLV-III. Polypeptides that includethese determinants can be synthesized, using organisms or cellsengineered by recombinant DNA techniques. The resulting polypeptides canbe recovered and included in a pharmaceutically acceptable carrier toinnoculate individuals to raise protection against HTLV-III infection.Specifically, the conserved epitopes of the env proteins of STLV-III andHTLV-IV are candidate immunogens for HTLV-III/LAV vaccine development.U.S. Ser. No. 06/790,830, filed Oct. 23 or 24, 1985 by Kennedy, Dreesmanand Essex, entitled Synthetic Peptides and Use for Diagnosis andVaccination for AIDS and ARC, is hereby incorporated by reference in itsentirety and discloses one method that could be used to derive a vaccinebased on polypeptide antigens of STLV-III or HTLV-IV.

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (a) a cell-surface glycoprotein of m.w. of about 160,000;(b) a cell-surface glycoprotein of m.w. of about 120,000; (c) a gagprotein of m.w. of about 55,000; (d) a gag protein of m.w. of about24,000; and (e) a glycoprotein of m.w. of about 32,000; wherein thepolypeptide has said molecular weight when produced by a cell infectedwith a strain of an HIV-2 virus, wherein the cell infected with a strainof the HIV-2 virus is a cell deposited as ATCC VR2129, and wherein thepolypeptide is not an HIV-1 polypeptide having the same molecular weightas the isolated polypeptide.
 2. The polypeptide of claim 1, wherein saidinfected cell is a progeny of one of the cell lines deposited as ATCCVR2129.
 3. A cell lysate comprising a cell-surface glycoprotein of m.w.of about 160,000 that has an antigenic determinant which, in common withan antigenic determinant of a protein produced by a cell infected with astrain of an HIV-2 virus, will react with a same given antibody, whereinthe cell infected with a strain of an HIV-2 virus is a cell deposited asATCC VR2129, and wherein the glycoprotein is not an HIV-I polypeptidehaving a m.w. of about 160,000.
 4. The cell lysate of claim 3,comprising a cell-surface glycoprotein of m.w. of about 120,000 that hasan antigenic determinant which, in common with an antigenic determinantof a protein produced by a cell infected with a strain of an HIV-2virus, will react with a same given antibody, wherein the cell infectedwith a strain of an HIV-2 virus is a cell deposited as ATCC VR2129, andwherein the glycoprotein of m.w. of 120,000 is not an HIV-I polypeptidehaving a m.w. of about 120,000.
 5. The cell lysate of claim 4,comprising a gag protein of m.w. of about 55,000 that has an antigenicdeterminant which, in common with an antigenic determinant of a proteinproduced by a cell infected with a strain of a HIV-2 virus, will reactwith a same given antibody, wherein the cell infected with a strain ofan HIV-2 virus is a cell deposited as ATCC VR2129, and wherein the gagprotein of m.w. of about 55,000 is not an HIV-I polypeptide having am.w. of about 55,000.
 6. The cell lysate of claim 5, comprising a gagprotein of m.w. of about 24,000 that has an antigenic determinant which,in common with an antigenic determinant of a protein produced by a cellinfected with a strain of an HIV-2 virus, will react with a same givenantibody, wherein the cell infected with a strain of an HIV-2 virus is acell deposited as ATCC VR2129, and wherein the glycoprotein of m.w. ofabout 24,000 is not an HIV-I polypeptide having a m.w. of about 24,000.7. The cell lysate of claim 6, comprising a glycoprotein of m.w. ofabout 32,000 that has an antigenic determinant which, in common with anantigenic determinant of a protein produced by a cell infected with astrain of an HIV-2 virus, will react with a same given antibody, whereinthe cell infected with a strain of an HIV-2 virus is a cell deposited asATCC VR2129, and wherein the glycoprotein of m.w. of about 32,000 is notan HIV-I polypeptide having a m.w. of about 32,000.
 8. An isolatedcell-surfce glycoprotein of m.w. of about 160 kD that has an antigenicdeterminant which, in common with an antigenic determinant of a proteinalso having a m.w. of about 160 kD produced by a cell infected with astrain of an HIV-2 virus, will react with a same given antibody, whereinthe cell infected with a strain of an HIV-2 virus is a cell deposited asATCC VR2129, and wherein the glycoprotein is not a 160 kD protein ofHIV-I.
 9. An isolated cell-surface glycoprotein of m.w. of about 120 kDthat has an antigenic determinant which, in common with an antigenicdeterminant of a protein also having a m.w. of about 120 kD produced bya cell infected with a strain of an HIV-2 virus, will react with a samegiven antibody, wherein the cell infected with a strain of an HIV-2virus is a cell deposited as ATCC VR2129, and wherein the glycoproteinis not a 120 kD protein of HIV-I.
 10. An isolated gag protein of m.w. ofabout 55 kD that has an antigenic determinant which, in common with anantigenic determinant of a protein also havig a m.w. of about 55 kDproduced by a cell infected with a strain of an HIV-2 virus, will reactwith the same given antibody, wherein the cell infected with a strain ofan HIV-2 virus is a cell deposited as ATCC VR2129, and wherein the gagprotein is not a 55 kD protein of HIV-I.
 11. An isolated gag protein ofm.w. of about 24 kD that has an antigenic determinant which, in commonwith an antigenic determinant of a protein also havig a m.w. of about 24kD produced by a cell infected with a strain of an HIV-2 virus, willreact with the same given antibody, wherein the cell infected with astrain of an HIV-2 virus is a cell deposited as ATCC VR2129, and whereinthe gag protein is not a 24 kD protein of HIV-I.
 12. An isolatedglycoprotein of m.w. of about 32 kD that has an antigenic determinantwhich, in common with an antigenic determinant of a protein also havig am.w. of about 32 kD produced by a cell infected with a strain of anHIV-2 virus, will react with a same given antibody, wherein the cellinfected with a strain of an HIV-2 virus is a cell deposited as ATCCVR2129, and wherein the glyco protein is not a 32 kD protein of HIV-I.13. An isolated polypeptide of claim 1, wherein said polypeptide isselected from the group consisting of: (a) a gag protein of m.w. ofabout 55,000; and (b) a gag protein of m.w. of about 24,000.